This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A. Specific Aims 1. Thorough examination of EHD protein expression in the developing cochlea. 2. Generate mice with inducible cochlear specific deletion of EHD1, EHD3 and EHD4. (Ehd1 fl/fl;Ehd3 fl/fl;Ehd4 fl/fl mice with Atoh1 Cre) to examine the effect of EHD protein loss in the cochlea. B. Studies and Results Aim 1. EHD protein expression in the cochlea. Attempts to micro-dissect cochlea from mice of different ages were carried out during the initial months of the project. Under the guidance of Dr. Sonia Sanchez (Assistant Professor, Creighton School of Dental sciences) the initial experiments were aimed at increasing the ability to identify the anatomical landmarks in the inner ear. Participation in a workshop at Jackson Laboratories further helped in learning cochlear dissections. However, due to lack of prior experience in micro-dissections, considerable amount of time in the initial months were taken up for micro-dissections. Once basic skills in cochlear dissections were acquired, further effort was aimed at examining EHD protein expression in the Organ of Corti. As shown (Fig.1) all EHD proteins are expressed in the inner and outer hair cells in the cochlea in one month old mice, Ehd4 null cochlea were used as control for EHD4 staining, while Ehd1 and Ehd3 null cochlea were used as control for EHD1 and EHD3 staining respectively (data not shown). Current efforts are aimed at becoming proficient in micro-dissection and immunostaining of cochlea from newborn mice. C. Significance : The results are too preliminary and the experiments will need to be repeated with proper controls to determine if there are significant abnormalities in tamoxifen induced Ehd1 fl/fl;Ehd4 fl/fl;Atoh1 CreER positive cerebellum. D. Plans : Staining experiments will be carried out to examine the deletion of EHD1 and EHD4 in the cochlea. We will also examine if EHD2 and EHD3 show a compensatory increase in expression in the hair cells. In addition, we will also examine the stria for compensatory increase in EHD protein expression in single and double EHD mutant mice. E. Publications: 1. George M, Rainey MA, Naramura M, Foster KW, Holzapfel MS, et al. (2011) Renal Thrombotic Microangiopathy in Mice with Combined Deletion of Endocytic Recycling Regulators EHD3 and EHD4 PLoS ONE 6(3): e17838. doi:10.1371/journal.pone.0017838